(Poly)peptides which represent the epitopes of the human herpes virus type 8

ABSTRACT

The present invention relates to (poly)peptides, which are recognized by anti-HHV8 antibodies of HHV-8 infected patients, whereby these (poly)peptides are not naturally occurring HHV-8 proteins. The present invention further relates to polymers, comprising at least two identical or different peptides according to the invention as well as conjugates, comprising said peptides and/or polymers thereof. Furthermore, this invention provides mixtures, comprising said peptides and/or polymers thereof, which are used to detect anti-HHV-8 antibodies with high sensitivity and specificity. In addition, the present invention relates to a diagnostic kit, comprising said peptides, polymers and/or mixtures thereof, which can be used for the detection of anti-HHV-8 antibodies and for the diagnosis of an HHV-8 infection, respectively.

The present invention relates to (poly)peptides that are recognized byanti-HHV-8 antibodies of HHV-8 infected patients. By definition, these(poly)peptides shall not comprise naturally occurring HHV-8 proteins.The invention further relates to polymers containing two or more(identical or different) inventory peptides as well as conjugatescomprising the inventory peptides and/or polymers thereof. Further wedescribe mixtures comprising the inventory peptides and/or polymersthereof that are particularly suited for use in procedures to detectanti-HHV-8 antibodies with high sensitivity and specificity. Inaddition, the present invention relates to a diagnostic kit comprisingthe inventory peptides, polymers and/or mixtures thereof for thedetection of anti-HHV-8 antibodies or diagnosis of HHV-8 infection,respectively.

The human herpesvirus 8 (HHV-8), also known as Kaposi sarcoma associatedherpesvirus (KSHV) is the purported etiological agent of Kaposi'ssarcoma and certain B cell lymphomas. Kaposi's sarcoma (KS) is the mostfrequent tumor in AIDS patients, affecting 20-30% of all patients duringthe course of their HIV infection. In the U.S., the incidence of KS inthis risk group is 20,000fold higher than in the general population [1].In some geographic areas, e.g. in Mediterranean countries or in Africa,the incidence in the general population is significantly higher.

AIDS-associated KS is characterized by an aggressive course and highmortality whereas classical KS is generally relatively indolent andslowly progressing. Other forms of the disease are endemic KS thatpredominantly affects children and adolescents in Sub-Saharan Africa,and iatrogenic KS in inmnune suppressed transplant recipients.

KS was first described more than a hundred years ago as a relativelyrare tumor occurring predominantly in elderly men of Mediterranean orEast European origin [2]. The characteristic external manifestations ofKS are sharply defined, differently colored (purple, brown, violet orblack) nodular lesions of the skin, mostly on the extremities but alsoin oral mucosa and viscera. Histologically, these lesions consist oflong spindle-shaped cells, apparently of endothelial origin, as well asa number of other cell types like fibroblasts, neo-vascular structures,infiltrating leukocytes and extra-vasated red blood cells.

The production of VEGF (vascular endothelial growth factor) issignificantly increased in tumor tissue leading to continuedangiogenesis and, hence, an extreme vascularization. Already years ago,epidemiological analyses gave hints that an infectious agent might beinvolved in the development of KS [1, 3]. Using a new PCR technique,Chang and Moore isolated from KS biopsies DNAs from a hitherto unknownhuman γ-2 herpesvirus [4]. This virus was called KSHV (Kaposi sarcomaassociated herpesvirus) or HHV-8 (human herpesvirus 8), respectively.Its 140 kb genome has been recently cloned and sequenced [5].

Since then a series of publications has shown beyond reasonable doubtthat all forms of Kaposi's sarcoma are correlated in practically 100% ofall cases with the presence of HHV-8 in KS lesions [6-11]. Moreover, PCRdetection of HHV-8 is a prognostic marker for later development ofKaposi's sarcoma [12]. Our own work has shown that seroconversion toHHV-8 is detectable on the average already two years before the clinicalmanifestation of KS.

Most likely HHV-8 is also involved in the pathogenesis of certainlymphoproliferative disorders such as multicentric Castleman's disease(MCD) or primary effusion lymphoma [13, 14]. Some data also hint to acontribution of HHV-8 in interstitial pneumonia and encephalitis [15].Recently published work suggests that HHV-8 is correlated with multiplemyeloma [16]. Although several experimental results are compatible withthe hypothesis that HHV-8 may be involved in the pathogenesis of thisdisease [17-22], a causal relationship is not yet proven. In somegeographic regions such as Central Africa, HHV-8 appears to berelatively widespread in the general population [8, 23-25]. Despite ahigher incidence of KS in these countries, the presence of an infectionwith HHV-8 alone appears to be necessary but not sufficient for thedevelopment of this disease.

Based on the available viral DNA sequences, several diagnostic testshave been developed that allow a direct or indirect detection of HHV-8.With the help of a relatively simple test, the polymerase chainreaction, even minute quantities of viral DNA can be detected in bloodor tissue samples [14, 26-28]. However, for some medical analyses PCRtests are not sensitive enough.

Immunosuppressed organ transplant recipients also have an increased riskof developing Kaposi's sarcoma (up to 5% for kidney transplantrecipients); the number of cases varies greatly with geographic origin.In countries of the Near and Middle East, KS is the most common posttransplant tumor; worldwide it ranks third. In developed countries(Europe, U.S. or Japan) about 1 to 1.5 m people have an increased riskof getting KS. Blood products for these patients should therefore beunconditionally tested for anti-HHV-8 antibodies as a marker for apossible contamination with HHV-8 in order to keep their infection riskas small as possible. In a broader sense this also applies for othermore or less immunocompromised groups, e.g. patients under high dosechemotherapy, dialysis patients, elderly people, neonates etc.

KS predominantly affects people suffering from various forms ofimmunodeficiency, and is only poorly treatable by chemotherapy, surgeryor with ionizing radiation. Presently, there is neither a causativetreatment nor a final cure for this disease. One has to consider thatimmune deficient individuals are exposed to an extraordinary risk todevelop Kaposi's sarcoma if they receive HHV-8 contaminated bloodproducts.

For routine diagnostics purposes one mostly uses ELISAs (enzyme-linkedimmunosorbent assays) because these tests are cheap and suitable forhigh throughput testing. The detection of HHV-8 specific antibodiesindicates prior contact with HHV-8 antigens. This antibody detection canbe positive even when the amount of viral DNA (in blood) is below thelevel of detection in a PCR analysis. Except for ELISAs [29-31], thepresence of HHV-8 specific antibodies can also be detected by indirectimmunofluorescence assays (IFAs) [25] or Western blots [32, 33].

However, the test procedures that have been described so far are eithervery time consuming (IFA or Western blot) or not sensitive enough (PCRfrom blood samples, ELISAs using previously described antigens.

The underlying technical problem for this invention was therefore todefine reagents the use of which allows a very sensitive and specificdetection of anti-HHV-8 antibodies.

This technical problem is solved by presenting the embodiments detailedin the claims section.

Thus the present invention relates to a (poly)peptide, which isrecognized by anti-HHV-8 antibodies in HHV-8 infected patients and ischaracterized as follows:

(a) it comprises one of the amino acid sequences presented in SEQ IDnos. 1 to 8;

(b) consists of one of the amino acid sequences presented in SEQ ID nos.1 to 8 or

(c) consists of an amino acid sequence which differs from one of theamino acid sequences described in (a) or (b) by one or severalsubstitutions, deletions and/or insertions; wherein the (poly)peptide isnot a naturally occurring HHV-8 protein.

Naturally occurring HHV-8 proteins in the sense of this invention areHHV-8 proteins that have the full-length amino acid sequence and are notdegraded. Usually or preferentially, the (poly)peptide is recognized byanti-HHV-8 antibodies in bodily fluids of HHV-8 infected individuals.Methods for the determination of the antigenic property of the(poly)peptide related to the invention as well as methods for theisolation of anti-HHV-8 antibodies, preferably from bodily fluids ofHHV-8 infected patients are known to those skilled in the art. Suchmethods comprise for example ELISA tests.

Bodily fluids in the sense of the present invention encompass e.g.blood, serum, plasma, lymph fluid, tissue fluid and extracts, e.g. frommucosa of respiratory, urogenital or gastro-intestinal origin. Further,this expression comprises bodily fluids that have been pretreated to beusable for the above mentioned analysis methods. Examples for this aresera diluted in vitro or treated with preservative agents forcryoprotection (e.g. glycerol) or coagulation inhibition (e.g. heparin).

The term “(poly)peptide” as used in relation to the present inventioncomprises both peptides and polypeptides. (Poly)peptides related to theinvention, which comprise one or more of the amino acid sequencescharacterized in SEQ ID no. 1 to 8 can be flanked by another HHV-8sequence as well as by a sequence unrelated to HHV-8. As alreadymentioned above, said (poly)peptide is not a naturally occurring HHV-8protein, i.e. said (poly)peptide is not a full length HHV-8 protein ascoded by HHV-8 genomes.

Preferably, said (poly)peptides are linear epitopes of different codingregions of the HHV-8 genome, and are less than 50 amino acids in length.More preferred is a length of maximal 15 amino acids, and most preferredis a length of the (poly)peptides related to the invention of 10 to 12amino acids.

The term “substitution” in the sense of the present invention comprisesboth conservative and non-conservative exchanges of amino acids.Conservative exchanges are those in which a neutral, hydrophobic, polar,basic or acidic amino acid is replaced by an amino acid of the sameclass. The different classes of amino acids, the classification of whichis determined by specific side chains is well known to those skilled inthe art. The crucial point is that the three-dimensional structure ofthe (poly)peptides is not perturbed in such a way that the peptide is nolonger recognized by anti-HHV-8 antibodies.

The peptides related to the invention were identified in a screening ofabout 3000 peptides according to the following criteria:

(a) minimal homology to peptides of known proteins, in particular tocorresponding epitopes of related herpesviruses as for instance EBV andCMV, and

(b) maximal antigenicity (by computer prediction)

A preselection of potentially immunodominant HHV-8 specific peptidesequences was achieved by an advantageous combination of differentcomputer programs (BLASTP, ANTIGENIC and BLASTALIGN) of the GeneticsComputer Group (GCG), Wisconsin.

All peptides selected by this procedure (292) were then individuallytested with a serum pool from KS patients and several control pools fromhealthy individuals.

The peptides that had passed this initial screen were then analyzed withseveral hundred sera from KS patients and controls with specialattention given to cross-reactions with EBV-, CMV- or HSV-specificantibodies. By these multiple tests, the peptides according to theinvention were identified that showed the desired reaction profile.

The (poly)peptides also comprise compounds that are produced bypeptidomimetics, preferably (poly)peptides resembling the abovementioned (poly)peptides in their immunological and diagnosticproperties. It is known in the art how such molecules can be designedand produced using L- and D-amino acids (e.g. Banerjee, Biopolymers 39(1996), 769-777). Preferably, the present invention relates to a peptidemix consisting of a combination of said (poly)peptides.

In a preferred embodiment, the peptide mix comprises the (poly)peptidesdisclosed in SEQ ID nos. 1, 3, 4 and 8.

In an especially preferred embodiment the peptide mix related to theinvention contains these (poly)peptides in a molar ratio of 1:1:1:1.

Using the peptide mix according to the invention, anti-HHV-8 antibodiescan be detected in an especially advantageous way. Surprisingly, thepeptide mix surpasses by far both the sensitivity and the specificity oftraditional detection methods (see below).

In another preferred embodiment, the (poly)peptide comprising an aminoacid sequence that differs from the amino acid sequences shown in (a) byone or more substitutions, deletions and/or insertions is a naturallyoccurring sequence variant.

Such peptides that represent sequence variants of the correspondingpositions in the genome of HHV-8 (5) may further increase thesensitivity of the detection. The specificity remains unaltered as theselection of peptides by minimal homology to known (viral) proteinsexcludes highly conserved regions to start with.

In another embodiment, the present invention concerns a polymercomprising at least two identical or different said (poly)peptides .

In the sense of the present invention, such polymers comprisehomopolymers consisting of several copies of a single (poly)peptide aswell as heteropolymers of any combination of said (poly)peptides wherebysuch heteropolymers may also contain several copies of one said(poly)peptide.

In a preferred embodiment, the polymer related to the invention ischaracterized by an unbranched chain of polymerized said (poly)peptides.

In another preferred embodiment, said (poly)peptide is characterized bybranched chains of polymerized said (poly)peptides.

Polymers with branched chains of polymerized (poly)peptides are producedby connecting peptides via an amino acid or an amino acid analog thatpossess two amino groups and one carboxyl group each capable of formingpeptide bonds. Such procedures are known in the art.

In another preferred embodiment, the present invention concerns polymersthat comprise peptides with the amino acid sequences disclosed in SEQ IDnos. 1, 3, 4 and 8.

In another preferred embodiment, said (poly)peptide is chemicallysynthesized.

Procedures for chemical synthesis of peptides using peptide synthesizersare known in the art. Preferably, the present invention relates tomixtures consisting of the polymers according to the invention.

In another embodiment, the present invention relates to a fusion proteincomprising a (poly)peptide or polymer according to the invention.

In another embodiment, the present invention concerns a polynucleotidecoding for a (poly)-peptide, polymer or fusion protein according to theinvention.

In a preferred embodiment, the polynucleotide is DNA or RNA.

The present invention further relates to a vector comprising apolynucleotide according to the invention.

In a preferred embodiment, the vector according to the invention is anexpression vector.

The term “expression vector” in the sense of the present inventioncomprises both prokaryotic and eukaryotic expression vectors. Thenecessary regulatory elements for the expression of a (poly)peptide areknown in the art and can be selected to achieve the desired expression.The term “expression” means transcription as well as transcription andtranslation. In particular, regulatory elements comprise promoters. Forexpression in prokaryotic cells of a poly-nucleotide according to theinvention a series of suitable promoters exist, e.g. the E. coli lac ortrp promoter, the lambda phage P_(R)- or P_(L)-promoter lacI, lacZ, T3,T7, gpt etc. Eukaryotic promoters include for instance the CMV immediateearly promoter, the HSV promoter, the thymidine kinase promoter, theSV40 promoter, LTRs of retroviruses or the mouse metallothioneinlpromoter. A multitude of expression vectors has been described forexpression in prokaryotic as well as eukaryotic cells, e.g. foreukaryotes pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) or GEM1(Promega Biotech, Madison, Wis., USA), pSV2CAT, pOG44 and forprokaryotes pQE70, pQE60, pBluescript SK, etc. In addition to promoters,vectors according to the invention may also contain other elements thatfurther increase the transcription, e.g. so-called enhancers. Examplesare the SV40 enhancer, the polyoma enhancer, the cytomegalovirus earlypromoter-enhancer and the adenovirus enhancer.

In another embodiment, the present invention relates to a host cellcontaining an expression vector according to the invention.

The term “host cell” according to the invention comprises prokaryotic aswell as eukaryotic host cells. Preferred prokaryotic host cells includee.g. E. coli cells, Streptomyces, Bacillus or Salmonella cells,preferred eukaryotic host cells include yeast cells, in particularSaccharomyces cerevisiae cells, insect cells as e.g. Drosophila or SF9cells, animal cells as e.g. CHO or COS cells, plant cells or mammaliancells.

In another embodiment, the present invention relates to a procedure forproduction of a (poly)peptide, polymer or fusion protein according tothe invention comprising the following steps:

(a) cultivation of a host cell according to the invention underconditions that foster the expression of the (poly)peptide, polymer orfusion protein; or

(b) in vitro transcription and/or translation of the polynucleotideaccording to the invention;

and isolation of the (poly)peptide, polymer or fusion protein producedas under (a) or (b).

The conditions for expression of a (poly)peptide are known in the artand can be selected according to the host cell and the expression vectorused. The same is true for the isolation of the expression product. Whenan expression vector is used that leads to the secretion of thesynthesized (poly)peptide, the (poly)peptide is isolated from theculture supernatant. When expression takes place intracellularly, theexpression product is isolated from the host cell. Methods for in vitrotranscription and/or translation are well known in the art. Forinstance, such procedures may be performed using commercial kitsaccording to the instructions of the manufacturer.

Preferably, additional purification steps known in the art, e.g. columnchromatography, may be used for the isolation of said (poly)peptide byremoving impurities such as cellular proteins, nucleic acids orcomponents of the in vitro transcription/translation system.

In another embodiment, the present invention relates to a conjugatecomprising a (poly)peptide and/or polymer and/or fusion proteinaccording to the invention or a (poly)peptide and/or polymer and/orfusion protein that is produced by said procedure.

In another embodiment, the present invention concerns a compositioncomprising at least one (poly)peptide and/or polymer and/or fusionprotein according to the invention and a conjugate according to theinvention, where appropriate also a pharmaceutically compatible carrierand/or diluent.

In a preferred embodiment, the composition is a pharmaceutical.

In an especially preferred embodiment, the pharmaceutical is a vaccine.In this embodiment, the peptides are preferably coupled to a carrier,either individually or in combination. Examples for suitable carriersare polystyrene beads, streptavidin, BSA or KLH.

Examples for suitable pharmaceutically compatible carriers and/ordiluents are known in the art and comprise e.g. phosphate bufferedsodium chloride solutions, water, emulsions as for instance oilwateremulsions, various forms of surfactants and detergents, sterilesolutions etc. Pharmaceuticals comprising such carriers can beformulated according to known conventional methods. Thesepharmaceuticals may be administered to an individual in a practicaldosis, either orally or parenterally (e.g. intravenously,intraperitoneally, subcutaneously, intramuscularly, locally,intranasally, intrabronchially or intradermally or through a catheterinto an artery. The physician in charge according to clinical factorsdetermines the dosage and mode of administration. It is known in the artthat the dosage depends on various factors such as height, weight, bodysurface, age, sex or the general health of the patient but also on thecharacteristics of the administered pharmaceutical, the duration andmode of application as well as on other drugs that may be givensimultaneously. A typical dosis may be in a range of 0.001 and 1000 mgwhereby dosages below or above this range are conceivable, in particularin consideration of the above mentioned factors. With regularadministration of the composition according to the invention, the dosageshould generally fall in a range between 1 μg and 10 mg units per day.In case of an intravenous administration (which is not recommendedbecause of the danger of anaphylactic shock), the dosis should be in arange between 1 μg and 10 mg per kg of body weight per minute.

The composition of the invention can be administered locally orsystemically. Preparations for a parenteral administration comprisesterile aqueous or non-aqueous solutions, suspensions or emulsions.Examples for non-aqueous solvents are propylenglycol,poly-ethylenglycol, plant oils as e.g. olive oil and organic estercompounds as e.g. ethyloleate that are suitable for injections. Aqueouscarriers include water, alcohol-water solutions, emulsions, suspensions,salt solutions and buffered media. Parenteral carriers include sodiumchloride solutions, Ringer-dextrose, dextrose and sodium chloride,Ringer-lactate and bonded oils. Intravenous carriers include fluid,nutrient and electrolyte supplements (e.g. those based onRinger-dextrose). The composition according to the invention may alsocontain preservatives and other supplements such as antimicrobialcompounds, antioxidants, complexing agents and inert gases. Furthermore,depending on the intended use, the composition according to theinvention may contain compounds such as interleukins, growth factors,differentiation factors, interferons, chemotactic proteins orunspecifically immunomodulatory agents.

In another embodiment, the present invention relates to a procedure forthe detection of anti-HHV-8 antibodies comprising the following steps:

(a) contacting a biological sample with at least one of the(poly)peptides and/or polymer and/or fusion protein according to theinvention, a (poly)peptide and/or polymer and/or fusion protein asproduced by said method , and/or a conjugate according to the invention,under conditions that allow the binding of antibodies; and

(b) detection of the antibodies bound in step (a)

In addition to the detection of anti-HHV-8 antibodies, said procedure isuseful as prognostic marker for the eventual development of Kaposi'ssarcoma in HIV infected individuals or persons with other natural oriatrogenic immune defects.

In another preferred embodiment, this procedure uses a mixture of(poly)peptides as characterized by the amino acid sequences disclosed inSEQ ID nos. 1, 3, 4 and 8.

The main advantages of said method with respect to previously describedprocedures [25, 29-34] are on one hand the high sensitivity (>96% ofsera from KS patients are positive as opposed to most proceduresdescribed in the literature, which only achieve sensitivities between 35and 85%) and on the other hand a very good specificity andreproducibility (with the ELISA technique duplicate variations aregenerally less than 5%).

In particular, the use of a combination of different (poly)peptides fromdifferent open reading frames of the viral genome rather than individualrecombinant antigens such as ORF65 [30] or the Minor Capsid Protein [31]allows the detection of a wider spectrum of HHV-8 specific antibodies.This is especially important with sera from patients who alreadysuffered from a marked immunodeficiency when they were infected withHHV-8 because the antibody titers may be very low in these cases. Otherpeptide-based immunoassays that use a single peptide achieve a muchlower sensitivity [29]. In addition, it is known from otherherpesviruses that the use of a single epitope is not sufficient toobtain a diagnostically relevant recognition quota of at least 90% ofthe true positive sera.

In an especially preferred embodiment of this procedure, said(poly)peptides are chemically synthesized.

By using chemically synthesized peptides rather than recombinantantigens from prokaryotic expression systems or unpurified virallysates, respectively as employed in other described procedures, areaction with non-specific antibodies (e.g. against E. coli proteins oragainst proteins as e.g. EBV or CMV) is practically excluded. Inhitherto described procedures, the antigens have to be present at a highdegree of purity as even minute amounts of contaminating material (e.g.residual E. coli proteins) can lead to false positive results in manysamples. A significant advantage of said procedure is that it does notrequire such time-consuming and costly purification steps (as e.g. prioradsorption of the test sera to E. coli lysates).

Preferably, the (poly)peptides and/or polymers and/or fusion proteinsaccording to the invention are biotinylated for use in said procedure.The biotinylated components are then bound to a streptavidin-coatedsolid phase via the highly specific biotin-streptavidin interaction.This coupling procedure ensures an even coating even with mixtures ofsaid (poly)peptides that are different in their charge or theirhydrophobicity, respectively.

In another especially preferred embodiment of this procedure, themixture contains these (poly)peptides in an equimolar ratio.

In another preferred embodiment, said procedure is an enzyme-linkedimmunosorbent assay (ELISA), an immunodot assay, an immunobead assay, apassive hemagglutination assay (PHA) or a peptide-antibody-peptidesandwich assay.

In another preferred embodiment of said procedure, the biological sampleis a pretreated or untreated form of blood, serum, tissue extract,tissue fluid, cell culture supernatant or cell lysate. The term“pretreated form” as used in the sense of the present invention, thereader is redirected to the above definition of the term “bodily fluids”since the possibilities of pretreatment and preparation are alsoapplicable in this context.

In another preferred embodiment, said procedure comprises the additionalstep:

(c) detection of unspecific binding reactions in step (a) caused byother antibodies that may be present in the biological sample

In ELISA tests, additional antibodies may bind independently of theantigen or unspecifically to the surface of the wells of a microplate,thus causing a false positive signal. False positive results due tounspecific binding to the matrix can be safely detected and eliminatedby using individual blank wells (without specific antigen). This is ofimportance for screening of sera in blood banks in order to avoid thatdonor blood is unnecessarily discarded due to false positive results ofHHV-8 tests.

The present invention further relates to a kit, comprising:

(a) at least one (poly)peptide and/or polymer and/or fusion proteinaccording to the invention, a (poly)peptide and/or polymer and/or fusionprotein produced according to the invention, and/or a conjugateaccording to the invention whereby the components may be separated or inform of a mixture; and where appropriate

(b) a solid phase to which at least one (poly)peptide and/or polymerand/or fusion protein according to the invention, a (poly)peptide and/orpolymer and/or fusion protein produced according to the invention,and/or a conjugate according to the invention may be bound;

(c) a sample diluent; and/or

(d) a negative control; and/or

(e) a positive control; and/or

(f) a reporter molecule

The term “reporter molecule” in the sense of the present inventioncomprises enzymes as e.g. peroxidase or alkaline phosphatase (in thesecases the detection system may be based on a colorimetric reaction asthe conversion of ABTS by a peroxidase) as well as other enzymes,radioisotopes, fluorophores, bioluminescent or chemiluminescentmolecules and the like or conjugates with e.g. a secondary antibodyrecognizing e.g. human IgG or IgM antibodies.

Said kit can be used to detect anti-HHV-8 antibodies and to diagnoseHHV-8 infections of mammals, preferably in bodily fluids such as blood,serum, tissue extracts, tissue fluids, as well as in in vitro cellculture supernatants and cell lysates (as test reagents). For theintended use, any suitable immunoassay may be chosen, in particular anenzyme-linked immunosorbent assay (ELISA), an immunodot assay, animmunobead assay, a passive hemagglutination assay (PHA), apeptide-antibody-peptide sandwich assay or other methods known in theart.

In a preferred embodiment, the kit contains a mixture of the(poly)peptides consisting of or containing the amino acid sequencesdisclosed in SEQ ID nos. 1, 3, 4 and 8.

In another embodiment, the present invention relates to the use of atleast one (poly)peptide and/or polymer and/or fusion protein accordingto the invention, a (poly)peptide and/or polymer and/or fusion proteinproduced according to the invention for the production of apharmaceutical for treatment of HHV-8 associated diseases.

In a preferred embodiment of the use according to the invention, theHHV-8 associated diseases are Kaposi's sarcoma, multicentric Castleman'sdisease, primary effusion lymphoma, interstitial pneumonitis,encephalitis and multiple myeloma.

In another embodiment, the present invention concerns the use of atleast one (poly)peptide and/or polymer and/or fusion protein accordingto the invention, a (poly)peptide and/or polymer and/or fusion proteinproduced according to the invention, and/or conjugate according to theinvention for the production of antibodies.

The production of antibodies is well known in the art and comprises e.g.the immunization of an animal with an antigen whereby the antigen may becoupled to a carrier and/or given in combination with otherimmunostimulating substances (cf. e.g. Harlow and Lane, “Antibodies, alaboratory manual”, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, 1988). Other known methods include the screening of antibodylibraries with the “phage display” technology and the recombinantproduction of the desired antibody.

In another embodiment the present invention concerns the use of at leastone (poly)peptide and/or polymer and/or fusion protein according to theinvention, a (poly)peptide and/or polymer and/or fusion protein producedaccording to the invention, and/or conjugate according to the inventionfor the detection of anti-HHV-8 antibodies.

The references cited in the present description are herewith defined tobe part of the description.

FIGURE LEGENDS

FIG. 1:

The serum activity against the preferred peptide mix was investigatedwith HIV-positive/KS-positive and HIV-positive/KS-negative patients.Columns represent the mean reactivity, error bars the standard deviationfrom the mean reactivity of (i) positive sera, (ii) sera from patientswith a CD4 cell count of less than 50/μl, (iii) negative sera. The meanserum reactivity in patients with advanced disease stages issignificantly lower than in the total collective.

FIG. 2:

Serum reactivity (CD4 cell count) of an HIV-positive, KS-positivepatient were tested using the HHV-8 ELISA (FACS analysis) in aretrospective analysis. HHV-8 specific antibodies are detectable in theblood of this patient already 14 months before the clinical diagnosis ofKaposi's sarcoma. The CD4 cell counts are a measure for the relativeimmune deficiency of the patient.

FIG. 3:

Schematic representation of the development of the HHV-8 ELISA test

As shown in the following examples, said procedure generally showspositive results years before the clinical diagnosis of Kaposi'ssarcoma. Since presently various anti-Herpes drugs are being evaluatedas potential KS therapeutics, such substances (or other antiviralsubstances effective for the treatment of KS) could possibly be used ina preventive way as soon as an infection with HHV-8 is safely diagnosedwith the procedure/kit according to the invention.

The following examples illustrate the invention.

EXAMPLE 1 ELISA Test

For the HHV-8 peptide mix ELISA, N-terminally biotinylated peptides of14 amino acids length are used (10 amino acids of the correspondingantigens plus the tetrapeptide Ser-Gly-Ser-Gly as spacer). The stocksolution of the individual peptides (1 μmol/ml in 100% DMSO) was diluted1:400 in PBS/5% Tween-20 to obtain a working solution used for coatingmicro-plates pre-coated with streptavidin (Combiplate 8, Life Sciencesor MC, MicroCoat). Wells are coated with 100 μl working solution (250pmol, i.e. a tenfold molar excess over the available amount of boundstreptavidin) by incubating for 30 minutes at room temperature. Themicroplates were then washed twice with PBS/0.2% Tween-20 and wereblocked one hour at room temperature with PBS/5% Tween-20. Beforeincubation with test material (one hour at room temperature with e.g.1:100 dilutions of human serum), the plates were washed three times withPBS/0.2% Tween-20.

After incubation with the test material, the plates are washed fivetimes and are then incubated for one hour at room temperature with a1:10,000 dilution of a peroxidase-coupled anti-human IgG or IgMantibody. Thereafter, the plates are again washed five times.

To detect bound conjugate, 200 μl of a 1 mg/ml ABTS solution inperoxidase working solution (Boehringer Mannheim) are added per well,and the plates are incubated 30 minutes at room temperature. Theabsorption of the wells is measured at 405 nm against a referencewavelength 492 nm. A sample is considered positive when the measuredvalue is higher than 200 mOD above the mean of the control value(without the peptide mix); 200 mOD correspond to at least 4 standarddeviations from the mean.

EXAMPLE 2 KS Serum Panel

To verify the serological efficiency, a panel of 380 individual serafrom 185 patients with Kaposi's sarcoma was analyzed. Of 256 serawithdrawn after KS diagnosis, 246 were positive with the preferredmixture 1 (peptides no. 1, 3, 4 and 8), which corresponds to asensitivity of 96.1%. Since this panel includes a good number of serafrom HIV patients whose immune system was severely damaged (a conditionin which the activation of antibody producing cells is limited), thesensitivity of the test with other panels is presumably even higher.

79 of 96 sera of HIV patients with strongly diminished immune functions(CD4 counts below 50) were positive with the described peptide mix; themean absorption at a wavelength of 405 nm was 0.342 with a standarddeviation of 0.264. For negative sera, the mean absorption was 0.046,and the standard deviation 0.038. 120 sera showed strong immune reaction(O.D._(405nm)>0.6). For these sera, the mean absorption was 1.15±0.49O.D._(405nm) (FIG. 1)

EXAMPLE 3 Assay Standardization

To standardize the assay, various plates, blocking methods and diluentswere tested. Reproducible coating quality and optimal reactionconditions crucially affect both the sensitivity and the reproducibilityof serologic assays. The described. HHV-8. ELISA shows very small intra-and inter-assay variations when the preferred peptide mix is used. Themean coefficient of variation of 79 duplicate determinations of serawith a low reactivity was 4.39%. In patients for whom consecutive serawere available, there was no detectable difference in the results ofsuccessive serum samples after the first positive sample. Blank values(incubation of the sera in wells without peptides) are very wellreproducible. The mean blank value from 30 plates was 128±45 milliOD

EXAMPLE 4 Control Panels

Various control panels as well as control peptides were used to test thespecificity of the HHV-8 antibody ELISA. Using an EBV control peptide(cf. table 2), analyses were carried out to compare antibody titersagainst HHV-8 and EBV (a closely related herpesvirus). In 56 out of 64cases the observed antibody titers were at least 30% divergent; the EBVseroprevalence was 86% with HIV-positive patients and 72% with thecontrols. 28 of 99 sera showed a high reactivity against both HHV-8 andEBV (OD_(405nm)>0.6), 39 of 99 sera had high anti-HHV-8 and low anti-EBVtiters, 7 out of 99 had low anti-HHV-8 and high anti-EBV titers, and 25out of 99 sera showed low reactivity against both HHV-8 and EBV antigens(OD_(405nm)<0.6). It can be concluded from these data that there was nosignificant cross-reactivity of the assay with EBV antigens.

EXAMPLE 5 Sensitivity

A highly reactive individual serum was tested in successive twofolddilutions (from 1:50 to 1:12,800). In the calorimetric assay, even thehighest dilution yielded an OD_(405nm)>1.0. The bandwidth of the assayis limited in this case by the relatively small useable window of thecolorimetric detection system. Using more sophisticated detectionmethods (e.g. chemiluminescence), linear quantitative determinations ofthe antibody titers should be possible over a range of more than threedecades. Thus, one may both quantitate high titered sera and safelyrecognize low antibody titers.

EXAMPLE 6 Prognostic Value

Of 20 KS patients who had been receiving medical treatment over a longerperiod of time, successive serum samples were analyzed for immunereactivity of HHV-8 specific antibodies. The first positive sample hadbeen withdrawn on the average 20.4±14.6 months prior to the clinicaldiagnosis. As in most cases the first available serum sample was alreadytested positive, the real incubation time is probably longer. The ELISAcould give hints at an existing risk for the development of a Kaposi'ssarcoma. The time course for one of these long-term patients is shown inFIG. 2.

EXAMPLE 7 Exclusion of False Positive Test Results

Of a total of 589 tested KS and control sera, 8 (1.36%) displayedreactivity with the empty wells of the microplates (pre-coated withstreptavidin). All these sera were negative in the HHV-8 ELISA afterpre-incubation in plates without peptide. Such false positive values canbe safely excluded by sample-specific blank values. None of thepreviously described test systems employs such individual blank values,which is why false positive tests resulting from unspecifically reactivesera cannot be recognized. This additional control prevents theunnecessary exclusion of blood samples and is therefore particularlyinteresting for blood banks from an economic point of view. Aspreliminary tests have shown, the proportion of unspecifically reactivesera appears to be much higher in panels from South European and Africancountries.

EXAMPLE 8 Comparison of Serologic Tests with DNA-based Detection Methods

Of 100 HIV infected test persons, both serum samples and genomic DNAfrom unfractionated peripheral blood cells were prepared. Sera wereanalyzed in the HHV-8 peptide mix ELISA, whereas the HHV-8 specific DNAwas determined in a polymerase chain reaction (PCR) with a detectionlimit of 5-10 copies/reaction (=1000-2000 copies/ml blood). In 5% ofthese cases, the serologic test was positive although the controlled PCRdetection remained negative.

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TABLE 1 Peptides found to be reactive with sera from KS patients.Position numbers relate to the published sequence of the HHV-8 genome byRusso et al. (5)(Genbank accession No. U75698)(SEQ ID NOS 1-8,respectively, in order of appearance). No. Seq ID Position Sequence ORF1 KS20A 35573- M Y E V F T D F P V tegument 35544 2 KS29bG 50000-D P A Y T N N T E A packaging 49971 protein 3 KS29bP 49394-R H M Y K P I S P Q packaging 49365 protein 4 KS65A 112443-M S N F K V R D P V capsid 112414 5 KS65X 111960- A R K P P S G K K Kcapsid 111931 6 KS73J 124726- Q E E Q E L E E V E LANA 124697 7 KSK12D118023- A I P P L V C L L A kaposin 117994 8 KS8.1A 75999-P T Y R S H L G F W Q E glycoprotein 76034

TABLE 2 Sequences of the control peptides used. Position numbers relateto HIV-1 HXB2 (Genbank accession no. M38432), Epstein-Barr virus strainB95-8 (V01555) or poliovirus type Sabin 1 (V01150). The first 4 aminoacids in italics (SGSG) (portion of SEQ ID NO: 9) in each sequence wereintroduced as a spacer (SEQ ID NOS 9-11, respectively, in order ofappearance). Seq No. ID Position Sequence ORF 1 HIV 8007-8036S G S G I W G C S G K L I C TM B (gp41) 2 EBV 100695-100724S G S G Q E P P A P Q A P I EBNA A 6 3 polio 2603-2632S G S G P A L T A V E T G A VP 1 B capsid

11 1 10 PRT Human herpesvirus 1 Met Tyr Glu Val Phe Thr Asp Phe Pro Val1 5 10 2 10 PRT Human herpesvirus 2 Asp Pro Ala Tyr Thr Asn Asn Thr GluAla 1 5 10 3 10 PRT Human herpesvirus 3 Arg His Met Tyr Lys Pro Ile SerPro Gln 1 5 10 4 10 PRT Human herpesvirus 4 Met Ser Asn Phe Lys Val ArgAsp Pro Val 1 5 10 5 10 PRT Human herpesvirus 5 Ala Arg Lys Pro Pro SerGly Lys Lys Lys 1 5 10 6 10 PRT Human herpesvirus 6 Gln Glu Glu Gln GluLeu Glu Glu Val Glu 1 5 10 7 10 PRT Human herpesvirus 7 Ala Ile Pro ProLeu Val Cys Leu Leu Ala 1 5 10 8 12 PRT Human herpesvirus 8 Pro Thr TyrArg Ser His Leu Gly Phe Trp Gln Glu 1 5 10 9 14 PRT Humanimmunodeficiency virus 9 Ser Gly Ser Gly Ile Trp Gly Cys Ser Gly Lys LeuIle Cys 1 5 10 10 14 PRT Epstein-Barr virus 10 Ser Gly Ser Gly Gln GluPro Pro Ala Pro Gln Ala Pro Ile 1 5 10 11 14 PRT Poliovirus 11 Ser GlySer Gly Pro Ala Leu Thr Ala Val Glu Thr Gly Ala 1 5 10

What is claimed is:
 1. A peptide mixture consisting essentially of amixture of peptides consisting of the amino acid sequences of SEQ IDNos.: 5 and 8, wherein either peptide is not a full length HHV-8 proteinas coded by the HHV-8 genome and the peptide mixture is recognized byanti-HHV-8 antibodies of HHV-8-infected patients.
 2. The peptide mixtureof claim 1, in which at least one peptide is chemically synthesized. 3.A method for the production of a peptide of the peptide mixture of claim1 comprising: (a) culturing a host cell comprising a polynucleotideencoding at least one peptide of claim 1 under conditions that result inthe expression of the peptide; or (b) transcribing and translating atleast one of said polynucleotides in vitro; and after completion of (a)or (b); (c) isolating the peptide made by (a) or (b).
 4. A compositioncomprising a peptide mixture of claim
 1. 5. The composition of claim 4,which is a pharmaceutical composition.
 6. A method for the detection ofanti-HHV8 antibodies, comprising: (a) contacting a biological samplewith the peptide mixture of claim 1 under conditions which allow thebinding of antibodies to said composition; and (b) detecting theantibodies bound in step (a).
 7. The method of claim 6, wherein saidmethod is selected from the group consisting of an enzyme-linkedimmunoabsorbent assay (ELISA), an enzyme immunodot assay, an immunobeadassay, a passive hemagglutination assay (PHA) and apeptide-antibody-peptide sandwich assay.
 8. The method of claim 6,wherein the biological sample is selected from the group consisting ofblood, serum, tissue extract, tissue fluid, cell culture supernatant anda cell lysate.
 9. A kit comprising at least one container comprisingindividual components or a mixture of components, wherein saidcomponents comprise: (a) the peptide mixture of claim 1, and optionallyat least one of the following: (b) a solid phase, to which at least oneof said components may be bound; (c) a sample diluent; (d) a negativecontrol; (e) a positive control; and (f) a reporter molecule.
 10. Thepeptide mixture of claim 1, wherein one or both petides is biotinylated.